Abstract TNF binding to its receptor TNFR1 triggers cell survival or cell death but the mechanisms that determine which of these two divergent responses is triggered remain to be fully elucidated. One cell death checkpoint is the activation of NF-kappaB transcription factors and subsequent induction of anti-apoptotic genes to inhibit the apoptosis pathway. Preliminary studies performed demonstrated the existence of another cell death checkpoint regulated by lysine 63-linked ubiquitination of RIP1. Ubiquitination of lysine 377 on RIP1 inhibits TNF-induced apoptosis first through an NF-kappaB-independent mechanism, and subsequently through an NF-kappaB- dependent mechanism. In contrast, in the absence of ubiquitination, RIP1 serves as a pro- apoptotic signaling molecule. Thus, RIP1 is a dual-function molecule that can be either pro-survival or pro-death depending on its ubiquitination state and thus serves as an NF- kappaB-independent cell death switch early in TNF signaling. This study proposes to examine how this cell death checkpoint may be regulated. (1) The mechanism by which ubiquitination of RIP1, or the lack of it, regulates the activation of caspases will be examined. (2) The contribution by RIP1-binding partners to this checkpoint will also be tested. (3) Animal models with genetic modifications in this novel cell death checkpoint will be developed to study the relevance of this checkpoint in immune function. Insights gained from these studies may lead to the development of pharmacological agents that can modulate this cell death checkpoint. Such agents may have therapeutic potential in inflammatory diseases where TNF plays a role.